T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

• MeSH
• aminokyselinové motivy MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• cytosol metabolismus   MeSH
• difuze MeSH
• fluorescenční spektrometrie MeSH
• fosforylace MeSH
• Jurkat buňky MeSH
• lidé MeSH
• optogenetika MeSH
• receptory antigenů T-buněk chemie   metabolismus   MeSH
• shluková analýza MeSH
• signální transdukce * MeSH
• světlo MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty metabolismus   MeSH
• vápník metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.

• MeSH
• benzoxaziny chemie   MeSH
• buněčná membrána chemie   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• fluorescenční spektrometrie MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• membránové lipidy chemie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Supercritical angle fluorescence (SAF) detection combines the axial discrimination and exquisite signal-to-noise ratio of total internal reflection fluorescence (TIRF) with the lateral discrimination and convenience of confocal excitation. This combination makes SAF ideal for fluorescence correlation spectroscopy (FCS) on membranes and other structures in close proximity to the coverslip. Here we report a straightforward modification of a commercial microscope to implement SAF FCS and demonstrate in both model supported lipid bilayers and cellular systems that this approach shows an increase in signal from membrane-bound fluorophores relative to fluorophores in solution, benchmarked against line-scanning FCS. SAF FCS allowed us to demonstrate that activation of the T cell receptor resulted in the recruitment of the kinase Lck to the plasma membrane as well as a reduction in Lck mobility within the membrane.

• Publikační typ
• časopisecké články MeSH